An nhr-85::GFP::AID*::3xFLAG knock-in allele for investigation of molting and oscillatory gene regulation

C. elegans NHR-85 is a poorly characterized nuclear hormone receptor transcription factor with an emerging role in regulating microRNA expression to control developmental timing. We generated the first NHR-85 translational fusion by knocking a GFP::AID*::3xFLAG cassette into the endogenous locus to tag all known isoforms. nhr-85 ::GFP::AID*::3xFLAG animals have wild-type broodsizes and NHR-85 ::GFP peaks in expression at the start of the L4 stage in epithelial cells. NHR-85 is not expressed in the germline, suggesting that while it might cooperate with the NHR-23 transcription factor to control microRNA expression, NHR-23 promotes spermatogenesis independent of NHR-85 . This nhr-85 ::GFP::AID*::3xFLAG strain will be a valuable resource for studying when and where NHR-85 acts to promote developmental timing.


Description
Molting is the process by which animals generate a new exoskeleton and shed their old one.Nematodes have a collagenous exoskeleton (cuticle) that is replaced at the end of each of four larval stages (Lažetić & Fay, 2017).The shedding and replacement of this cuticle is thought to be coordinated by a recently discovered, large-scale genetic oscillation in which ~20% of genes peak one time during each larval stage (Hendriks et al., 2014;Meeuse et al., 2020;Tsiairis & Großhans, 2021).The homologs of several mammalian circadian rhythm regulators such as Per and RORα (LIN-42 and NHR-23 in C. elegans) regulate C. elegans molting (Jeon et al., 1999;Kostrouchova et al., 1998Kostrouchova et al., , 2001;;Monsalve et al., 2011).NHR-85, the C. elegans homolog of another mammalian circadian rhythm regulator (Rev-ERBα), is a poorly-characterized nuclear hormone receptor (NHR) transcription factor implicated in molting (Gissendanner et al., 2004).To gain insight into endogenous NHR-85 expression we used CRISPR/Cas9-mediated genome editing to insert a GFP::AID*::3xFLAG tag into the 3' end of the gene to produce a C-terminal translational fusion to all predicted isoforms (Figure 1A).nhr-85 RNAi was reported to cause an egg-laying defect (Gissendanner et al., 2004).To test whether the tag disrupted NHR-85 function, we monitored nhr-85::GFP::AID*::3xFLAG egg laying in a broodsize assay and found that the strain had wild-type fecundity with no obvious egg-laying defect (Figure 1B) .
Our nhr-85::GFP::AID*::3xFLAG strain has been used to analyze how NHR-85 cooperates with the NHR-23 transcription factor to control the expression of the lin-4 microRNA (Kinney et al., 2023).We were able to reproduce the peak in expression of NHR-85::GFP in vulval precursor cells at L4.0 and L4.9 (Figure 1C,D).We also observed the reported expression in hypodermal cells as well as in seam cells (Figure 1D).We confirmed that the expression was specific to the nhr-85::GFP::AID*::3xFLAG strain, as no nuclear GFP signal was observed in wild-type animals (Figure 1E).NHR-85::GFP expression is nuclear and excluded from nucleoli (Figure 1C,D).Interestingly, we did not observe NHR-85::GFP expression in the L4 or adult germline (Figure 1F), and nhr-85 null animals are viable and fertile (Kinney et al., 2023).NHR-23 is expressed in the L4 and male germline and promotes spermatogenesis (Ragle et al., 2020(Ragle et al., , 2022)).These data suggest that while NHR-23 and NHR-85 cooperate in the soma to regulate gene expression, nhr-23 has an nhr-85-independent role in regulating spermatogenesis.Future use of this strain should allow for conditional, tissue-specific depletion to gain insight into when and where NHR-85 acts to promote oscillatory gene expression.

C. elegans strains and culture
C. elegans strains (see table in Reagents) were cultured as originally described (Brenner, 1974), except worms were grown on MYOB instead of NGM.MYOB was made as previously described (Church et al., 1995).Animals were cultured at 20°C for all assays, unless otherwise indicated.For general strain propagation, animals were grown at 15°C according to standard protocols.Brood sizes were performed as previously described (Ragle et al., 2022).

Microscopy
Imaging was performed as previously described (Johnson et al., 2023).Animals were synchronized by alkaline bleaching and released on MYOB before harvesting at the indicated developmental timepoints.Animals were picked into a 15 µl drop of M9+5 mM levamisole on a glass slide with a 2% agarose pad and secured with a coverslip.Animals were imaged using a Plan-Apochromat 63×/1.4Oil DIC lens on an AxioImager M2 microscope (Carl Zeiss Microscopy) equipped with a Colibri 7 LED light source and an Axiocam 506 mono camera.We used Fiji software (version: 2.0.0-rc-69/1.52p) to process images (Schindelin et al., 2012).For the comparisons in the developmental timecourse or between strains, we set the exposure conditions to avoid pixel saturation of the brightest sample and kept equivalent exposure for imaging of the other samples.

Figure 1 .
Figure 1.NHR-85::GFP is expressed in epithelial cells but not the germline: (A) Schematic of the nhr-85 gene with location of the endogenous GFP::AID*::3xFLAG knock-in.Black rectangles are coding exons, gray rectangles are the 5' and 3' untranslated regions, and the arrow indicates the direction of the gene and position of the introns.(B) Brood size analysis of wild type and nhr-85::GFP::AID*::3xFLAG animals.n=11 for N2 (WT)